Summary and Abstract: High Relaxivity PSMA-Targeted Contrast Agents for MRI of Prostate Cancer The goal of this proposal is to test the hypothesis that compact, high-relaxivity targeted contrast agents (HR-TCAs) that selectively bind to prostate-specific membrane antigen (PSMA) will dramatically improve magnetic resonance imaging (MRI) of prostate cancer (PCa). With high expression in cancerous cells and low expression in non-cancer tissue, PSMA is an excellent biomarker for imaging PCa including sites of metastasis. A peptidomimetic urea inhibitor, DCL, has shown high affinity for PSMA leading to a variety of near-infrared (NIR) dye agents for fluorescence and photoacoustic imaging, 18F agents for PET, and low-relaxivity gadolinium (Gd) agents for MRI of PCa. To date, low molecular weight, HR-TCAs for MRI of PCa have not been described. In order to test this hypothesis a new, modular method for the synthesis of compact peptide-based TCAs will be employed to synthesize mono and di Gd multimers of PSMA-directed contrast agents, using the high-relaxivity Gd-chelate, Gd- DO3A. The design embraces the advantages of peptides in bioavailability and clearance, and the enhanced safety of closed-cage macrocyclic Gd agents. Targeted HR-TCAs will require far lower concentrations of Gd agent versus low-relaxivity and non-targeted agents, further increasing their safety. To evaluate contrast efficiency in MRI, the T1 relaxation time will be measured by nuclear magnetic resonance (NMR) using a method developed at RIT. Cell binding of TCAs to PSMA+ and PSMA- cells in-vitro will be measured by AA spectroscopy. A dual modal NIR- MRI analog, also available by the modular method, will be evaluated using confocal fluorescence microscopy (CFM) to quantitate binding to PCa cells and study enhancement by endocytosis as a highly effective means of amplifying signal intensity as observed in our preliminary results. To ensure clinical safety, the stability of Gd chelation will be assessed by accelerated de-metalation studies using an HPLC-MS assay developed at RIT. The best TCAs will be translated for in-vivo testing by collaborators at Roswell Park Cancer Institute (RPCI), who will also mentor RIT undergraduates working at RPCI in summers. Mouse xenograft models will be used to test the hypothesis that MRI can clearly identify human PCa following systemic administration of PSMA-directed TCAs. The testing will include targeting PCa cells that have metastasized. We will examine toxicity in mice followed by PK/PD studies by assaying for the TCA in excretions and tissues by ICP-MS. We expect the small, peptide-based TCAs will maintain excellent bioavailability, stability and clearance in-vivo. A key aspect of this proposal is the involvement of four undergraduates from RIT, two of whom will also conduct summer research at RPCI. The collaboration highlights expertise in synthesis, cell and tissue biology and MRI agent chemistry at RIT, and in cancer biology and pre-clinical MRI at RPCI. These combine to provide a vibrant learning environment in cancer imaging research for undergraduates, with the achievable goal of transforming MRI of PCa. The development of safe, small-peptide based, PSMA targeted high-relaxivity contrast agents will transform imaging of PCa and provide a breakthrough for accurate diagnosis and treatment, leading to positive outcomes in PCa patients.